“Zernike Phase Plate TEM for Cryo-Tomography and Single Particle Data Acquisition and Analysis”
Barbara Armbruster – JEOL USA, Inc.
Phase contrast light microscopy has long been an indispensible tool for live-cell imaging, but this effective technique has only recently been successfully applied to transmission electron microscopy. A Zernike phase plate consists of an amorphous carbon film of defined thickness with a small central hole. At the backfocal position, the zero-order beam passes through the central hole while electrons traversing the thin film are phase-shifted by π/2. The optimal design for phase contrast cryo-electron tomography would include a field-emission gun, a three lens condenser system with independent adjustment for each lens, and an objective focal length of 5mm or larger to accommodate the phase plate holder plus a room-temperature aperture to serve as a heat shield. The small size of the central hole of the phase plate requires accurate software control to center parallel illumination in all low dose modes and tilt angles. Zero-loss energy filtering roughly doubles the signal-to-noise ratio and is essential for removing inelastic scattering when imaging thick samples. The realization of this design should extend the practical examination of the proteome to include nanomachines as small as 100kDa.