When collecting data using a two-condenser lens TEM, it is imperative that the lenses are set to illuminate the sample with a maximally parallel beam in order to prevent changes in defocus from causing magnification changes. Larger defocus changes produce a greater degree of magnification distortion. Although the magnification changes resulting from non-parallel illumination are subtle, they will nonetheless degrade the high-resolution information of a reconstruction when thousands of particles from different defoci are averaged together.
This protocol assumes that: 1) Gun alignments have already been performed on the microscope (note that only service engineers should change the gun settings on an X-FEG), 2) the Microscope User Interface and Digital Micrograph software are installed on the scope and camera computers, respectively, 3) grids amenable to data collection have already been prepared and loaded into the microscope, and 4) SA-mode magnifications use NanoProbe mode, all M and LM magnifications use MicroProbe.