Continuous electron diffraction tomography data collection with Gatan counted cameras

Continuous electron diffraction tomography, also known as microcrystal electron diffraction (MicroED) or three-dimensional electron diffraction (3DED), allows researchers to determine the structure of proteins and small molecules at atomic resolution. This technique uses an electron beam to obtain diffraction patterns from small specimen crystals (>500 nm), which can then be used to reconstruct the 3D structure of the molecule. Here we will describe the steps required to collect high-quality diffraction tomography data from K3® and Metro® cameras. 

Camera setup for microED/3DED data collection

  1. Reduce the beam's intensity by changing the spot size to one of the highest numbers (smallest spot) available. 
  2. Start the camera Live view and change the camera length so the SA aperture is entirely within the field of view.
  3. Ensure that the desired crystal is located and remains within the selected area aperture as you tilt to the maximum and minimum desired tilt range.
  4. Decrease the beam intensity until fewer than 250,000 e-/s (K3) or 500,000 e-/s (Metro) reach the camera. The total electron dose rate can be measured using a script published on the Gatan website or manually calculated using the built-in analysis tools in DigitalMicrograph® software.
  5. Switch to the Diffraction mode and center the undeflected diffraction beam on the camera. 
  6. Using the Focus knob, adjust the central bright spot to a minimum diameter while viewing the pattern on camera. These conditions result in saturated pixels displaying contrast reversal of the central diffraction beam. 
  7. If quantification of diffraction peak intensity is desired, keeping the brightest Bragg intensities below 40 e-/pix/s (K3) or 80 e-/pix/s (Metro) is essential.
  8. Adjust the exposure time based on the stage tilt speed, the tilt range, and the total target dose on the specimen. Typically, data is collected at 2 – 40 subframes per second with a rotation speed ranging from 0.1 to 2 degrees per second.
  9. Click Capture.