Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) provide sub-nanometer visualizations of cells and atomic-scale reconstructions of key biological molecules like proteins. However, traditional defocus-based techniques suffer from limited contrast that reduces the signal-to-noise ratio of images. Phase plates for electron microscopy convert the phase shifts imparted by a sample into amplitude contrast that may be directly detected by a camera and thus promise to make optimal use of information from weak phase objects. We have demonstrated a laser phase plate where a continuous laser in a Fabry-Perot build-up cavity reaches an intensity of approximately 400 GW/cm 2 and is used to coherently manipulate the electron beam for optimal phase contrast. On a modified FEI Titan 80–300 microscope, we have demonstrated in-focus phase contrast imaging of biological specimens with a contrast transfer function that is unity up to the limit set by spherical aberrations. Even with defocus applied, the improved contrast from the phase plate at low spatial frequencies is useful for identifying sample heterogeneity. Reconstructions of test proteins including apoferritin and RuBisCO show that laser phase plate images retain high-resolution information up to limits imposed by chromatic aberrations. We have recently built and installed a laser phase plate on a modified fourth-generation Krios microscope equipped with a cold-FEG, CEOS spherical aberration corrector, energy filter, and phase plate module. The integrated system has successfully produced high-resolution images of specimens ranging from microtubules to hemoglobin, and we are currently investigating the phase plate’s utility for imaging small proteins and performing cryo-electron tomography.
Cooper et al. (Sun,) studied this question.