Influence of electron dose rate on electron counting images recorded with the K2 camera

Abstract

A recent technological breakthrough in electron cryomicroscopy (cryoEM) is the development of direct electron detection cameras for data acquisition. By bypassing the traditional phosphor scintillator and fiber optic coupling, these cameras have greatly enhanced sensitivity and detective quantum efficiency (DQE). Of the three currently available commercial cameras, the Gatan K2 Summit was designed specifically for counting individual electron events. Counting further enhances the DQE, allows for practical doubling of detector resolution and eliminates noise arising from the variable deposition of energy by each primary electron. While counting has many advantages, undercounting of electrons happens when more than one electron strikes the same area of the detector within the analog readout period (coincidence loss), which influences image quality. In this work, we characterized the K2 Summit in electron counting mode, and studied the relationship of dose rate and coincidence loss and its influence on the quality of counted images. We found that coincidence loss reduces low frequency amplitudes but has no significant influence on the signal-to-noise ratio of the recorded image. It also has little influence on high frequency signals. Images of frozen hydrated archaeal 20S proteasome (~700 kDa, D7 symmetry) recorded at the optimal dose rate retained both high-resolution signal and low-resolution contrast and enabled calculating a 3.6 Å three-dimensional reconstruction from only 10,000 particles.

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

“…Virtually all high-resolution cryoEM reconstructions reported to date have been obtained using high-end microscopes operated at 300 kV, such as the FEI Titan Krios (Amunts et al, 2014; Fernandez et al, 2014; Lu et al, 2014; Voorhees et al, 2014; Wong et al, 2014; Zhang et al, 2013a; Zhang et al, 2013b), the FEI F30 Polara (Allegretti et al, 2014; Bai et al, 2013; Cao et al, 2013; Li et al, 2013a; Li et al, 2013b; Liao et al, 2013; Wolf et al, 2010; Zhang et al, 2008) or the JEOL JEM3200FSC (Baker et al, 2013; Cong et al, 2010). 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.…”

Practical aspects of diffractive imaging using an atomic-scale coherent electron probe

“…Synthesizing conventional STEM images is a good starting point for further data analysis, allowing the quality of the 4D datasets to be evaluated. Questions on the sensitivity [17] and quantum efficiency [18] have been explored for pixel direct detection cameras, but their use for absolute-scale quantitative imaging in STEM has received less attention. Contrast mismatch or "Stobbs factor" issues in TEM have been discussed since the 1990s [19].…”

“…It should be emphasized that the analysis above is the fundamental limit based on the statistics of detecting quantum particles. Current systems are likely to have lower SNR due to their detection quantum efficiency [18] and the readout and Landau noise, though the latter may be avoided or significantly reduced by using a counting mode [17] or a thicker detector design [41]. Note too that the outer angle limit determined from the observable intensity variation also depends on the scattering strength of the objects (elements, probe position, orientation, etc.…”

Single-particle cryo-EM data acquisition by using direct electron detection camera

“…Centroiding quadruples the total number of pixel in an image and extends the Nyquist limit to twice of the Nyquist limit defined by the physical pixel size. The drawback of a counting camera is that it requires a relatively low electron dose rate on the camera to minimize the effect of coincidence loss, i.e., when two primary electron events overlap, only one event is counted and the other is lost, which causes certain image artifacts [16].…”