Biological Cryo-EM

DE cameras offer ultimate resolution, sensitivity, and the largest available field-of-view to optimize low-dose applications such as single particle cryo-EM and cryo-tomography. Collect high-quality data quickly, use our powerful movie-mode processing algorithms, and generate stunning and reliable 3D reconstructions.

The Impact of Direct Detection for Biological Cryo-EM

High-Resolution EMDB Depositions

Number of structures deposited in the EMDataBank (http://www.emdatabank.org/), grouped by reported resolution. Data was downloaded from the EMDB on May 18, 2015. The first published cryo-EM structure from a direct detector was GroEL on a DE-12 Camera System from Bridget Carragher and Clint Potter at The Scripps Research Institute.

Since the introduction in 2008 of the DE-12 Camera as the first commercially-available direct detection TEM camera, the field of biological cryo-EM seen an explosion of near-atomic resolution structures. Direct detection cameras have not only dramatically improved data quality, but they have also changed the entire paradigm of cryo-EM data collection:

  • Dramatically higher signal-to-noise ratio in each image means particles are easier to align and classify, and fewer particles are needed for reconstructions.
  • Signal all the way up to Nyquist frequency enables imaging at lower magnifications to increase the number of particles per image.
  • Movie-mode acquisition (intrinsic dose fractionation) enables motion correction of stage drift and beam-induced motion.
  • Movie-mode acquisition enable high-dose imaging of beam sensitive specimens, as “damage compensation” algorithms can be used to maximize the signal-to-noise ratio at every spatial frequency, even beyond traditional radiation damage limits.

Breaking the 3 Å Barrier

Sub-3 Å Resolution 3D Reconstruction

Cryo-EM density map and model of a variant of AAV (adeno-associated virus). The resolution of the full map was measured to be ~2.9 Å resolution. Data was collected on a DE-20 Camera mounted on a Titan Krios, running Leginon for automated data acquisition (which took ~3 days). Courtesy of Scott Stagg, Florida State University.

Our DE-Series Cameras enable the most efficient imaging conditions for any high-resolution cryo-EM. While some direct detectors require ultra-long exposure times to acquire decent images, our cameras deliver the best image in seconds. Not only does this speed up data collection, it also makes motion correction more effective because users can fractionate their dose in much shorter time increments.

Productivity is further boosted by our large field-of-view. For example, the DE-20 is nearly 40% larger than another direct detector on the market, and our true 8k×8k DE-64 is nearly 5x larger! A large field-of-view saves time and money by giving you more particles per image (which means you need to collect many fewer images). Furthermore, a large field-of-view yields better integrated signal across the entire frame to improve CTF correction and further boost the resolution of your 3D reconstructions.

The proof is our customers’ results. The combination of performance, throughput, and flexibility from our cameras have enabled our customers to generate many single particle cryo-EM reconstructions at near-atomic resolution, including the 2.9 Å structure shown at the left.

Perhaps even more important than the absolute resolution (FSC value), reconstructions from DE cameras yield reliable, reproducible, de novo 3D reconstructions and atomic models. In fact, the atomic models from density maps generated from DE cameras have exceeded the quality of X-ray crystal structures of the same specimens (see Wang, et al., Nature Communications 5 (2014), 4808).

Intermediate Resolution Cryo-EM

Structure of the AcrAB-TolC Multidrug Efflux Pump

Cryo-EM images and a pseudoatomic model of the drug efflux pump (771 kDa). Data was collected on a DE-12 Camera System operating at 25 fps mounted on a JEOL 3200FSC (300 kV) at 20,000x magnification and 50 e2 total exposure. Raw frames were processed using motion correction and damage compensation. (a) A representative raw image of the purified pump. White circles indicate particles with long axis almost normal to the viewing plane; black circles show particles with the long axis parallel to the viewing plane. Scale bar is 50nm. (b) The reconstructed map and pseudo-atomic model. (c) A slice through the reconstruction and model, with the inner membrane (IM) and outer membrane (OM) labeled. Published in Du, et al., Nature 509 (2014), 512-515.

While the recent burst of ultra-high-resolution structures has garnered the most attention, the vast majority of biological questions addressed by cryo-EM remain answerable at much lower resolutions. In many experiments, generating an atomic model is either unnecessary (especially if the atomic model of the components have already been solved by other methods) or impractical (for example, when there is significant heterogeneity). Often, reliably determining the secondary, tertiary, and/or quaternary structure is sufficient.

Our DE-Series Cameras deliver the largest-available field-of-view together with the excellent image quality and movie-mode corrections of direct detection. Since our cameras are designed to work optimally at nearly any beam brightness, you can collect images at low magnifications (for an even bigger field-of-view) without worrying about degrading image quality. This means highly-efficient imaging: lots of particles with little effort. In this highly competitive field, high throughput and productivity are absolutely crucial.

Cryo-Tomography

HIV core pseudotypes with ASLV envelop Infected CV-1 cells

A Z-slice through a cryo-tomography reconstruction of HIV core pseudotypes with ASLV envelop Infected CV-1 cells. Data was collected on a DE-20 Camera System at 200 kV with motion correction for each tilt-series image. The video below shows z-slices through a 3D reconstruction of another similar tomogram of the same specimen. Courtesy of Greg Melikian, Mariana Marin, Cheri Hampton, and Elizabeth Wright at Emory University, Department of Pediatrics.

The high sensitivity and resolution of our DE-Series Cameras is also perfectly suited for tomography, where the total electron dose must be fractionated into a large number of very low-dose tilt-series images. Despite the low dose in each tilt image, our DE-Series Cameras allow users to visualize Thon rings to do CTF fitting in each tilt image. Furthermore, the exceptional contrast from our cameras produce stunning tomograms so you can clearly visualize the detail you need to see for your experiments.

Tomography also demands the largest possible field-of-view. One of the challenges of cellular tomography (including correlative light and electron microscopy, or CLEM) is precisely locating and tracking the exact region-of-interest throughout a whole tilt series. Having a large camera (such as our 8k x 8k DE-64) ensures that your region-of-interest is actually recorded in the tilt series. Additionally, a large field-of-view provides a much wider cellular context, allowing you to see surrounding effects and relationships that would have been missed with a smaller camera. After all, won’t the discoveries from your tomography experiments be limited if you can only capture a tiny fraction of each cell?