Vibration-free Cryo-electron Tomography using Ultrafast Pulses of Electrons

Cryogenic electron tomography in a transmission electron microscope (cryoET in a TEM) is a powerful tool in biology, delivering high-resolution structural information for objects in situ inside frozen-hydrated cells. Currently, the technique’s potential is limited by two factors. The first is low throughput. Subtomogram averaging requires images of tens to hundreds of thousands of particles at various tilt angles, prohibitive for anything but a few ideal cases. The second is reduced image quality. Tilted images are almost always of poorer quality than untilted images of the same particle. It has been suggested that an important contribution to the tilt-induced blurring of images is drumhead-like trembling of the sample, with an amplitude of oscillation normal to the grid. This oscillation is invisible to the electron beam when the sample is untilted, but gradually becomes more problematic at the higher tilt angles required for cryoET. We propose a proof-of-principle investigation and demonstration of the cryoET method using new innovations in TEM instrumentation: namely, the laser-free ultrafast electron pulser. This device is capable of producing pulse trains of electrons at Hz to GHz repetition rates without degrading the electron beam quality. The final deliverable of this Project is a dataset quantifying the performance of our prototype in improving the spatial resolution of high-tilt-angle cryoET images. In Phase I, we will build the drive electronics and test them on a conventional TEM at Brookhaven National Laboratory using an existing ultrafast pulser retrofitted to the instrument. These tests are cost-effective using a cryo-tilt holder. In Phase II, we will refine the design for commercial production and install and test it on a full cryo-tomography TEM. Higher resolution imaging of biomolecules beyond the state of the art is of immense interest. Our technology will push the state of the art in cryo-electron microscopy and will generate significant scientific and societal benefits. 3D tomographic imaging of smaller proteins and cellular structures holds direct promise to advance our understanding in diverse fields from biology to medicine. Examples include new insights into cellular function and new drug development. We also may obtain faster cryoET throughput by imaging faster than acoustic vibrations, eliminating the long vibration-damping wait after each motorized tilt-angle adjustment.