An imaging filter to enable high-resolution cryogenic electron tomography (cryo-ET) at York

Advances in cryogenic electron microscopy (cryo-EM) allow imaging and the computational reconstruction of biological molecules at sufficient resolution to see atomic-level detail. Because unstained biological material is sensitive to damage by electrons, imaging must be done at low dose (fluence) and is therefore noisy. To get to high-resolution, thousands of homogeneous particle images are averaged together. Because of these limitations, image quality is critical and the invention of 'direct electron detectors' that detect electrons with great sensitivity ('detective quantum efficiency'), was a major advance. A further enabling technology is the use of imaging filters, which improve contrast by removing inelastically scattered electrons that are nonproductive for imaging. Pairing a direct electron detector with an imaging filter provides a powerful combination for electron imaging: the filter removes the nonproductive 'inelastically scattered' electrons that cloud the image and the detector captures the productive 'elastically scattered' electrons with high sensitivity. Together, they create a cutting-edge system that allows the high-resolution imaging of thicker samples, enabling the transition from studying molecules of interest as purified complexes to directly imaging them in their natural cellular environment.

The Research the Equipment will enable
Here we apply for a Selectris imaging filter to upgrade the York Glacios cryogenic electron microscope (EM) to enhance the imaging capabilities of the already installed Falcon-IV direct electron detector. The imaging filter improves the imaging of all samples, but is particularly advantageous for the thicker specimens used in cryogenic tomography (cryo-ET). In cryo-ET, intact biological material is tilted in the microscope and the images are computationally reconstructed to provide a 3D map of the object. Cryo-ET allows macromolecular structural analysis within the complex environment of the cell and of pleomorphic objects that lack regular structure, thereby enabling new levels of biological insight. The filter will also aid imaging demanding targets purified from cells that are either small (<<100 kDa) or flexible.