“Building Proteins in a Day: Efficient 3D Reconstruction for Cryo-EM”

Wednesday, March 18, 2015

“Building Proteins in a Day: Efficient 3D Reconstruction for Cryo-EM”

Ben Bammes, Direct Electron

Transmission electron microscopy (TEM) is a powerful technique for visualizing structure at nanometer or Angstrom resolution. However, multiple factors reduce the resolution and signal-to-noise ratio (SNR) of TEM images. These include the microscope instrumentation, dynamic specimen processes (e.g. drift, beam-induced motion, charging, radiation damage, etc.), and inefficient electron detectors. Direct detection cameras have overcome many of these obstacles, dramatically boosting the resolution and efficiency of TEMs for biology, and in addition to improved detection efficiency and resolution, the architecture of direct detection cameras allows for fast, continuous streaming with no dead time between consecutive frames (known as “movie-mode”). Several studies have already shown how movies can be exploited to correct stage drift and beam-induced specimen motion, however movies also provide intrinsic dose fractionation, which allows microscopists to choose their image exposure ex post facto by using subsets of frames from each movie. Users may also exploit movie-mode imaging with “damage compensation,” which applies a low-pass filter to each movie frame based on the expected radiation damage at the corresponding cumulative specimen dose. Using this method allows microscopists to collect data with exposures beyond the conventional radiation damage limit of their specimen, which yields a very high contrast without sacrificing resolution.

This talk will introduce the DE-20 Camera System (which is now installed on the JEOL 3200FSC at NYSBC), discuss the best practices for data collection and image processing with the DE-20’s movie-mode, and share recent results from Direct Electron’s cameras.