Microfabricated porous TEM grids for improved phase contrast and CryoEM imaging
DESCRIPTION (provided by applicant): Cryo electron microscopy is a powerful technique for generating 3-D images of macromolecules and their interactions with fine structure within cells. These materials are imbedded in their native state within a thin layer of amorphous ice for imaging in a transmission electron microscope. By taking a series of images at various angles, 3-D reconstructions can be made. However, these low-density materials produce little contrast in TEM images, and this effect is compoundedby the limited electron current that can be applied to these fragile samples before damage is likely to occur. One of the most promising methods to increase image clarity is through the use of phase contrast imaging where specially designed phase plates (Zernike phase plates in this work) are used to develop contrast between electrons that are scattered by the sample and those that pass directly through. Unfortunately, despite the availability of sophisticated microscopes that are designed to accept phaseplates, researchers are frustrated by the lack of consistently manufactured, high-quality phase plates. In this proposed project, extensive microfabrication expertise in the manufacture of ultrathin materials for EM grid applications is being applied to the problem of phase plate production. Through a collaboration between TEMWindows.com, a respected ultrathin membrane fabricator, and the Wadsworth Center, a pioneer in the development of cryo and phase contrast electron imaging, a series of phase platedesigns will be produced using well-controlled and manufacturable methods, and these devices will be directly compared to current carbon-based phase plates. The goal is to produce stable, consistent, and low cost phase plates that show little backgroundcharging over long duration in a TEM. PUBLIC HEALTH RELEVANCE: The project described in this proposal will provide a commercial source of contrast enhancing phase plates to remove a bottleneck and advance the development of cryo electron microscopy.This technique is used to study the 3-D structure of large molecules and how they interact with the complex structures within cells. This structural information is critical to understanding fundamental processes involved in various disease states that impact public health.
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