Cryo-EM Sample Handling

The Nobel prize in chemistry recently awarded to Dubochet, Frank, and Henderson, for developing cryo-electron microscopy methods for high resolution structure determination in biomolecules is a testament to cryo-electron microscopy’s importance. While cryo-electron microscopy resolution, imaging cameras, and data analysis methods have developed considerably over the years, the preparation and handling of suitable cryogenic thin samples lags. Thus, this proposed work aims in improving cryogenic sample handling. To provide artifact-free cryo-electron microscopy samples, we propose to develop cryo- focused ion beam milled ex situ lift out sample handling and micromanipulation methods. Applying ex situ lift out to manipulate focused ion beam prepared samples will allow a fast, easy, reproducible, and cost-effective method compared to existing focused ion beam techniques. The proposed work will be broken down into 3 major milestones. Milestone 1 is to model heat transfer of proposed sample geometries and manufacture sample handling fixtures needed. Milestone 2 is to test FIB milling methods and geometry necessary for EXLO manipulation. Milestone 3 is to perform EXLO micromanipulation steps and test sample vitrification in an EM. Understanding the structure/property relationships of plant ultrastructure and biomolecules is essential for development of new energy resources, drugs, medical treatments, and treatment of human diseases. Anything that can be done to improve the time and efficiency to results can greatly enhance the human quality of life. Using the proposed methods, we estimate a savings of $100’s - $1,000’s per sample compared to existing sample preparation methods, while improving the time to results from days to hours.