Hardware Considerations to Optimize Zernike Phase Contrast TEM for Cryo-tomography and Single Particle Data Acquisition

Phase contrast TEM imaging with Zernike phase plates has been successfully applied to produce images of vitreously frozen specimens, including cryosections, whole cells and single particles, with optimum contrast at or near true focus [1-7]. Several column designs from 120-400kV have been modified to include a phase plate holder inserted in the back focal plane: Hosokawa et al [8] described a 120kV design incorporating an additional lens doublet and heated phase plate holder, Motoki et al [9] presented high resolution images of amorphous germanium and graphite taken on a 200kV platform with the phase plate positioned at a conjugate back focal plane, the 3.5nm structure of rat TRPV4 cation channel was reconstructed from images taken in a 300kV phase contrast TEM equipped with a liquid helium stage [10], and critical experiments to understand phase contrast optics and improve the quality of phase plates continue [5]. As discussed in Danev et Marko et al and Armbruster et al the optimal design for phase contrast single particle imaging and cryo-electron tomography requires a thorough review of all optical elements to increase image contrast and automation capabilities to maximize ease of use. Features required in a dedicated column configuration have been incorporated in the JEM-2200FS