FEI's direct electron detector developments: Embarking on a revolution in cryo-TEM.

In early 2011 FEI Company launched the "Falcon", its first commercial direct electron detector product intended for application in 3-D electron microscopy in the life sciences. In this paper we discuss the principle of direct electron detection and its implementation in Falcon cameras. We describe the signal formation in the sensor and its impact on the detection quantum efficiency (DQE) of the sensor. Insights into the signal formation led us to improved camera designs. Three significant improvements are discussed. (1) Back thinning of the sensor. This is implemented in the second-generation Falcon (Falcon 2), where the sensor thickness is reduced to 50 µm, and in the latest generation Falcon 3 detector with further back-thinning down to 30 µm. (2) The introduction of electron counting, a signal processing technology implemented in Falcon 3. (3) Dose fractionation mode, which allows the user to access intermediate results during the illumination of the sample.

A posteriori correction of camera characteristics from large image data sets.

Large datasets are emerging in many fields of image processing including: electron microscopy, light microscopy, medical X-ray imaging, astronomy, etc. Novel computer-controlled instrumentation facilitates the collection of very large datasets containing thousands of individual digital images. In single-particle cryogenic electron microscopy ("cryo-EM"), for example, large datasets are required for achieving quasi-atomic resolution structures of biological complexes. Based on the collected data alone, large datasets allow us to precisely determine the statistical properties of the imaging sensor on a pixel-by-pixel basis, independent of any "a priori" normalization routinely applied to the raw image data during collection ("flat field correction"). Our straightforward "a posteriori" correction yields clean linear images as can be verified by Fourier Ring Correlation (FRC), illustrating the statistical independence of the corrected images over all spatial frequencies. The image sensor characteristics can also be measured continuously and used for correcting upcoming images.

Molecular architecture of the "stressosome," a signal integration and transduction hub.

A commonly used strategy by microorganisms to survive multiple stresses involves a signal transduction cascade that increases the expression of stress-responsive genes. Stress signals can be integrated by a multiprotein signaling hub that responds to various signals to effect a single outcome. We obtained a medium-resolution cryo-electron microscopy reconstruction of the 1.8-megadalton "stressosome" from Bacillus subtilis. Fitting known crystal structures of components into this reconstruction gave a pseudoatomic structure, which had a virus capsid-like core with sensory extensions. We suggest that the different sensory extensions respond to different signals, whereas the conserved domains in the core integrate the varied signals. The architecture of the stressosome provides the potential for cooperativity, suggesting that the response could be tuned dependent on the magnitude of chemophysical insult.

Cryo-electron microscopy reveals a novel DNA-binding site on the MCM helicase.

The eukaryotic MCM2-7 complex is recruited at origins of replication during the G1 phase and acts as the main helicase at the replication fork during the S phase of the cell cycle. To characterize the interplay between the MCM helicase and DNA prior to the melting of the double helix, we determined the structure of an archaeal MCM orthologue bound to a 5.6-kb double-stranded DNA segment, using cryo-electron microscopy. DNA wraps around the N-terminal face of a single hexameric ring. This interaction requires a conformational change within the outer belt of the MCM N-terminal domain, exposing a previously unrecognized helix-turn-helix DNA-binding motif. Our findings provide novel insights into the role of the MCM complex during the initiation step of DNA replication.

Magnification variations due to illumination curvature and object defocus in transmission electron microscopy.

It has previously been shown that - in theory - magnification variations can occur in an imaging system as a function of defocus, depending on the field curvature of the illuminating system. We here present the results of practical experiments to verify this effect in the transmission electron microscope. We find that with illumination settings typically used in the electron microscopy of biological macromolecules, systematic variations in magnification of ~ 0.5% per microm defocus can easily occur. This work highlights the need for a magnification-invariant imaging mode to eliminate or to compensate for this effect.