Transformative advances in cryogenic electron microscopy (cryo-EM) have expanded our ability to visualize cellular and biomolecular structure at near atomic resolution. However, a significant hurdle to reaching the full potential of cryo-EM facilities, including those associated with the DOE, involves difficulties with sample preparation. The requirement to vitrify ultrathin samples, especially those associated with single particle analysis (SPA), makes it challenging to reliably produce and easily identify usable cryo-EM sample grids. This project will address these difficulties by developing a novel tool to rapidly screen cryo-EM grids and provide critical information on the thickness of the vitreous ice/sample layer. This information will enable real-time optimization of vitrification procedures, to increase the success and yield of sample production, and improve subsequent biomolecular structure determination using advanced electron microscopes. The technology being developed could be used in either standalone instruments or incorporated into full robotic vitrification systems. A proof-of-concept device will be designed and constructed to validate our approach for rapid and precise screening of cryo-EM sample grids. Its performance will be quantified using realistic vitrified samples on standard cryo-EM grids. Lateral spatial resolution will routinely be below 1?m, and ice-thickness measurement targeted for a precision of a few nm. Several different embodiments of the tool will be evaluated for planning the more sophisticated Phase II prototype instrument. Cryo-EM has experienced explosive growth in recent years, with projected worldwide annual revenue for just electron microscopes to exceed $1B before 2030. Improved sample preparation equipment represents a substantial percentage of this market sector. Cryo-EM data has become central for many areas of basic and applied science, including drug discovery in the huge biotech and pharma industries. This project seeks to develop a crucial tool for greatly improving the throughput of structures solved in many disciplines of life sciences, medicine, environmental research, and other fields requiring structure determination at the highest possible resolution.
