Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy

Abstract

We report on initial results of using a new Direct Detection Device (DDD) for single particle reconstruction of vitreous ice embedded specimens. Images were acquired on a Tecnai F20 at 200KeV and a nominal magnification of 29,000x. This camera has a significantly improved signal to noise ratio and modulation transfer function (MTF) at 200 KeV compared to a standard CCD camera installed on the same microscope. Control of the DDD has been integrated into Leginon, an automated data collection system. Using GroEL as a test specimen, we obtained images of ~30K particles with the CCD and the DDD from the same specimen sample using essentially identical imaging conditions. Comparison of the maps reconstructed from the CCD images and the DDD images demonstrates the improved performance of the DDD. We also obtained a 3D reconstruction from ~70K GroEL particles acquired using the DDD; the quality of the density map demonstrates the potential of this new recording device for cryoEM data acquisition.

Scanning transmission electron microscopy strain measurement from millisecond frames of a direct electron charge coupled device

“…This extends the scope of application for direct electron detectors, whose excellent detection quantum efficiency and signal-to-noise ratio have as yet enhanced nearly uniquely low-dose applications as used for biological imaging. [23][24][25][26][27] In this study, we used 300 keV electrons as the microscope performs best at this energy as to STEM probe formation. However, it can be expected that lowering the TEM acceleration voltage to less than 100 kV will lead to a strong improvement of the camera's point spread function, from which both SANBED and conventional TEM applications will profit.…”

Near-atomic resolution reconstructions using a mid-range electron microscope operated at 200kV

“…Pairing one of these microscopes with a direct detector clearly represents an ideal setup to study biological specimens at near-atomic resolution but the costs associated with the purchase and maintenance of this high-end equipment is a major undertaking for many universities and institutes worldwide. To follow-up on our previous studies aiming at characterizing the potential of mid-range electron microscopes when coupled to direct detectors (Campbell et al, 2012; Milazzo et al, 2011; Veesler et al, 2013), this contribution provides an assessment of the achievable resolution limits by single-particle cryoEM when using an FEI TF20 Twin electron microscope coupled to a Gatan K2 Summit camera. We demonstrate that it is possible to obtain cryoEM maps of sufficient quality for de novo modeling of protein structures of different sizes and symmetries when a widely used mid-range electron microscope is combined with a direct detector.…”

Cellular structural biology as revealed by cryo-electron tomography

“…In CCDs, the electrons first pass through a scintillator, where they are converted into photons, that then travel through a fiber-optic bundle before being recognized by the sensor; however, this causes information degradation, which is manifested at the high frequency ranges. The recent introduction of direct detector device (DDD) cameras (Li et al, 2013;Faruqi and McMullan, 2011;Milazzo et al, 2011;Ruskin et al, 2013;Bammes et al, 2012) is revolutionizing the field of cryo-EM and cryo-ET. In comparison to CCDs, DDDs have a complementary metal-oxide semiconductor (CMOS) chip that is able to directly recognize electrons.…”